New fluorescent adenosine A1-receptor agonists that allow quantification of ligand-receptor interactions in microdomains of single living cells.

Fluorescence spectroscopy is becoming a valuable addition to the array of techniques available for scrutinizing ligand-receptor interactions in biological systems. In particular, scanning confocal microscopy and fluorescence correlation spectroscopy (FCS) allow the noninvasive imaging and quantification of these interactions in single living cells. To address the emerging need for fluorescently labeled ligands to support these technologies, we have developed a series of red-emitting agonists for the human adenosine A1-receptor that, collectively, are N6-aminoalkyl derivatives of adenosine or adenosine 5'-N-ethyl carboxamide. The agonists, which incorporate the commercially available fluorophore BODIPY [630/650], retain potent and efficacious agonist activity, as demonstrated by their ability to inhibit cAMP accumulation in chinese hamster ovary cells expressing the human adenosine A1-receptor. Visualization and confirmation of ligand-receptor interactions at the cell membrane were accomplished using confocal microscopy, and their suitability for use in FCS was demonstrated by quantification of agonist binding in small areas of cell membrane.

Fluorophore-tagged GPCR ligands.

Fluorescently tagged drug molecules can be successfully employed to visualize the location of their receptor target at the single-cell level. Furthermore, if their binding to the receptor is reversible, one can now obtain detailed pharmacological information such as affinity using single-molecule detection techniques. When coupled to the growing exploitation of fluorescence-based read-outs in high throughput and high content screening, it is clear that fluorescent molecules offer a safer, more powerful and more versatile alternative to radioligands in molecular pharmacology and drug discovery. GPCR pharmacology has benefited enormously from the application of fluorescence-based technologies and we now possess a much greater understanding of this receptor family's basic molecular mechanisms of action through the careful design and judicious use of fluorescent peptide and small-molecule-based ligands.

Influence of mammographic screening on trends in breast-conserving surgery in Ireland.

Debate exists about the influence of screening on breast cancer surgery. We compared rates and odds of mastectomy and breast-conserving surgery (BCS) during 1994-1999 between Northern Ireland, which has had organized mammographic screening (age group 50-64) since 1993, and the Republic of Ireland, where large-scale screening did not begin until 2000. Trends in BCS were similar in both populations: significant increases in BCS rates for age groups 20-49 and, especially, 50-64, and significant increases in mastectomy rates for age group 65+ only. Use of BCS among surgical patients was significantly higher in Northern Ireland (46%) than the Republic of Ireland (35%) and, in Northern Ireland, was significantly higher in age group 50-64 than other age groups. Geographic and age-related variations were substantially, but not wholly, explained by tumour characteristics and screen-detection status. Among Northern Ireland women aged 50-64, BCS use among screen-detected cases, even after adjustment for tumour characteristics, was 40-50% higher and increased more rapidly during 1994-1999 than among other cases. However, trends in BCS in Northern Ireland were not explained by changes in screen-detection status or tumour characteristics. Our findings are consistent with expectations that mammographic screening, together with modern treatment guidelines, should lead to reduced use of mastectomy and increased (proportional) use of BCS. But similar trends occurred in the absence of large-scale screening (in the Republic of Ireland), and surgical treatment of screen-detected cases did not reflect tumour characteristics alone.

Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry.

A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 microm (100 x 2.1mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r(2)>0.99) over the range of 0.03-300 microM in urine and 0.03-15 microM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10-150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human ß-adrenoceptors.

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human ß-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity ß-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional ß2- and ß1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human ß2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent ß2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

Application of fluorescence correlation spectroscopy to the measurement of agonist binding to a G-protein coupled receptor at the single cell level.

The A1-adenosine receptor (A1-AR) is a member of the G-protein coupled receptor superfamily, which has significant pathophysiological importance in disorders such as heart arrhythmias, asthma and stroke. Here, we have used fluorescence correlation spectroscopy (FCS) to facilitate the study of A1-AR pharmacology at the subcellular level. To this end, we have successfully designed and synthesised a fluorescently labelled A1-AR agonist, ABA-BY630. ABA-BY630 is an N6- derivative of adenosine conjugated to the red-excited fluorophore, BODIPY" 630/650. In CHO cells expressing the human A1-AR, ABA-BY630 shows strong and potent agonist activity at this receptor. Specific binding of ABA-BY630 to the A1-AR in cell membranes of living CHO cells can also be visualised using confocal microscopy. Moreover, using FCS, we can detect and quantify the binding of ABA-BY630 to the A1-AR in a small area (0.2 microm2) of the upper cell membrane. FCS measurements indicate the presence of at least two populations of receptor-ABA-BY630 complexes with diffusion times of 8 and 233 ms. The quantity of both of these complexes was significantly reduced by pre-incubation with the A1-AR antagonist DPCPX. Application of FCS in conjunction with ABA-BY630 will allow the comparison of A1-AR pharmacology in single cells from healthy and diseased tissue.